Article pubs.acs.org/JAFC
Activity and mRNA Levels of Enzymes Involved in Hepatic Fatty Acid Synthesis in Rats Fed Naringenin Toru Hashimoto†,‡ and Takashi Ide*,§ †
Nippon Shinyaku Co., Ltd., 14 Nishinosho-Monguchi, Kisshoin, Minami-ku, Kyoto 601-8550, Japan Department of Food and Nutrition, Faculty of Human Life, Jumonji University, 2-1-28 Sugasawa, Niiza, Saitama 352-8510, Japan
§
ABSTRACT: We investigated the physiological activity of naringenin in affecting hepatic lipogenesis and serum and liver lipid levels in rats. Rats were fed diets containing 0, 1, or 2.5 g/kg naringenin for 15 d. Naringenin at a dietary level of 2.5 g/kg significantly decreased the activities and the mRNA levels of various lipogenic enzymes and sterol regulatory element binding protein-1c (SREBP-1c) mRNA level. The activities and the mRNA levels were also 9−22% and 12−38% lower, respectively, in rats fed a 1 g/kg naringenin diet than in the animals fed a naringenin-free diet, although the differences were not significant in many cases. Naringenin at 2.5 g/kg significantly lowered serum triacylglycerol, cholesterol, and phospholipid and hepatic triacylglycerol and cholesterol. This flavonoid at 1.0 g/kg also significantly lowered these parameters except for serum triacylglycerol. Naringenin levels in serum and liver dose-dependently increased, and hepatic concentrations reached levels that can affect various signaling pathways. KEYWORDS: naringenin, hepatic lipogenesis, serum lipid levels, liver lipid levels, rat
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INTRODUCTION Plants contain various kinds of flavonoids. Flavonoids are considered to be beneficial by preventing various diseases including cancer,1,2 arteriosclerosis,3−5 hypertension,6 diabetes,7,8 and the development of obesity.9,10 Some of their physiological activities can be ascribed to their antioxidant propensities.3,4,6−8 In addition, findings suggest that some flavonoids affect various signaling pathways and hence modulate gene expression and metabolic activities.2,7,9−12 This may also account for the physiological activities of flavonoids. Among the various plant phenolic compounds, studies have indicated that the citrus flavonoid naringenin lowers hepatic and serum lipid levels in experimental animals.13−22 With regard to the mechanisms underlying the lipid-lowering effect of this flavonoid, studies have indicated that naringenin affects various aspects of hepatic lipid metabolism.13−22 However, the observations were often inconclusive and conflicting. It has been well demonstrated that alteration in hepatic lipogenesis is critical for modifying the serum lipid concentration. In relation to this, Junget al.17 reported that a diet containing 2 g/kg naringenin significantly decreased the activities of fatty acid synthase and glucose 6-phosphate dehydrogenase in C57BL/KsJ-db/db mice. More recently, Mulvihill et al.19 showed that a diet containing 30 g/kg naringenin, but not a diet containing 10 g/kg of this flavonoid, relative to a diet free of this compound, significantly reduced the incorporation of intraperitoneally injected [14C]acetate into hepatic fatty acids accompanying the downregulation of sterol regulatory element binding protein-1c (SREBP-1c) mRNA levels in low-density-lipoprotein (LDL) receptor-null mice. In addition, naringenin at 30 g/kg lowered the incorporation of this tracer into hepatic cholesterol. However, the use of acetate as a tracer to estimate the rate of lipogenesis has long been criticized due to the possible divergent extents of the dilution of © 2015 American Chemical Society
the specific activity of the cytosolic acetyl-CoA pool available for fatty acid and cholesterol synthesis among different physiological conditions.23 Other information regarding the effect of naringenin on hepatic lipogenesis has been lacking. In addition, no studies have examined the effect of naringenin on hepatic lipogenesis in rats. In these contexts, we examined the effect of naringenin on hepatic lipogenesis in rats in the present study.
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MATERIALS AND METHODS
Animals and Diets. Male Sprague−Dawley rats obtained from Charles River Japan, Kanagawa, Japan, at 4 weeks of age were housed individually in animal cages in a room with controlled temperature (20−22 °C), humidity (55−65%), and lighting (lights on from 07:00 to 19:00 h), and fed a commercial nonpurified diet (Type NMF, Oriental Yeast Co., Tokyo, Japan). After 7 d of acclimatization, the rats were divided into 3 groups consisting of 7 animals each and fed purified experimental diets for 15 d. The experimental diets contained 0, 1, or 2.5 g/kg naringenin. Chemically synthesized naringenin was purchased from Tokyo Chemical Industry, Tokyo, Japan. The purity of naringenin was 95% (manufacturer’s statement). The basal composition of the experimental diet was as follows (in g/kg): palm oil, 100; casein, 200; corn starch, 150; cellulose, 20; mineral mixture, 35; vitamin mixture, 10; L-cystine, 3; choline bitartrate, 2.5; and sucrose to 1 kg. The compositions of vitamin and mineral mixtures were the same as those described by Reevs et al.24 Naringenin was added to the experimental diets in lieu of sucrose. Energy contents of experimental diets were 4,230, 4,226, and 4,220 kcal/kg for the naringenin-free diet and diets containing 1 and 2.5 g/kg naringenin, respectively. Protein, fat, and carbohydrate comprised 19.2, 21.3, and 59.5% of the total energy in the diets, respectively. We used palm oil as a dietary fat at a level of 100 g/kg. This oil is a saturated fat mainly Received: Revised: Accepted: Published: 9536
August 14, 2015 October 14, 2015 October 14, 2015 October 14, 2015 DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
Article
Journal of Agricultural and Food Chemistry composed of palmitic and oleic acids which causes higher hepatic lipogenesis and serum and hepatic triacylglycerol levels compared to unsaturated fat. Therefore, the experimental diets containing this oil are suited to test the physiological activity of food factors to lower hepatic lipogenesis and serum and tissue lipid levels. The dietary fat level (100 g/kg) employed is comparable to that observed in a typical Japanese diet (about 25% of energy). The rats were allowed free access to the diets and water throughout the experiment until the day they were killed. This study was approved by the review board of animal ethics of Jumonji University, and we followed the university’s guidelines in the care and use of laboratory animals. Enzyme Assays. Upon termination of the experiments, animals were killed by bleeding from the abdominal aorta under isoflurane anesthesia, after which livers were excised immediately. About 2.5 g of each liver was homogenized with 15 mL of 0.25 M sucrose containing 1 mM EDTA and 3 mM Tris-HCl (pH 7.2), and centrifuged at 200000g for 30 min. The activity of enzymes involved in fatty acid synthesis was measured spectrophotometrically using 200000g supernatant of the liver homogenate as detailed previously.25 The activities of enzymes involved in hepatic fatty acid oxidation were measured in total homogenate, as described previously.25−27 RNA Analyses. RNA in the liver was extracted, and mRNA abundance was analyzed by quantitative real-time PCR, as detailed elsewhere.27 The nucleotide sequences of primers and probes designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA) have been reported previously.28 mRNA abundance was calculated as the ratio to the β-actin level in each cDNA sample and is expressed as a fold change, setting a value of 1 for rats fed a diet free of naringenin. Analyses of Serum and Liver Lipids and Serum Insulin. Liver lipids were extracted, and triacylglycerol, phospholipid, and cholesterol concentrations in the extracts were determined, as described previously.29 Serum lipid concentrations were assayed using commercial enzyme kits (Wako Pure Chemical, Osaka, Japan). Serum insulin concentrations were analyzed with a commercial ELISA kit (Morinaga Co., Tokyo, Japan). Analyses of Serum and Liver Naringenin. The concentrations of naringenin in serum and liver were analyzed by HPLC according to methods described previously30,31 with some modifications. For the analysis in serum, 100 μL of 0.58 M acetic acid and 50 μL of βglucuronidase/sulfatase solution (131,100 units/mL) of Helix pomatia origin (Sigma-Aldrich Japan Co., Tokyo, Japan) were added to 1 mL of serum, and the mixture was incubated at 37 °C for 4 h. After enzymatic hydrolysis, each sample was diluted with 4 mL of 70 mM sodium dihydrogen phosphate and added to 6 μg of daidzein dissolved in 25 μL of methanol as an internal standard. The sample was loaded onto a Sep-Pak C18 cartridge (Waters, Milford, MA), and the cartridge was washed successively with 10 mL of 70 mM sodium dihydrogen phosphate and 1 mL of water. Flavonoids were then eluted with 6 mL of methanol. For the liver sample, 0.2 g of the tissue was added to 2 mL of 80% methanol (v/v) in water and 6 μg of daidzein dissolved in 25 μL of methanol. The liver sample was homogenized with a Polytron-type homogenizer. The homogenate was added to 4 mL of 25 mM citrate buffer (pH 5.0), and the mixture was rehomogenized. Next, the homogenate was added to 100 μL of βglucuronidase/sulfatase solution and incubated at 37 °C for 4 h. The hydrolysate was centrifuged at 12000g for 20 min. The supernatant was then collected in a glass tube, to which 10 mL of 70 mM sodium dihydrogen phosphate was added. The mixture was subjected to solidphase extraction, as described for the serum sample. Methanolic solutions of flavonoids obtained from serum and liver samples were evaporated to dryness under a stream of nitrogen, the residues were dissolved in 200 μL of methanol, and a 10 μL aliquot was analyzed by HPLC using a reversed-phase Capcell Pak AG120 C18 column (Shiseido, Tokyo, Japan) at a flow rate of 1.0 mL/min. The mobile phase consisted of 0.5% phosphoric acid in water (v/v) and acetonitrile (70:30, v/v). Naringenin and daidzein were detected using a spectrophotometer at a wavelength of 290 nm. Statistical Analyses. Microsoft Excel add-in software (Excel Statistics 2010, Social Survey Research Information Co., Tokyo, Japan)
was used for the statistical analysis. The constancy of the variance and normality of the distribution of the observations were evaluated by Levene’s test and the Kolmogorov−Smirnov test, respectively. If variances were heterogeneous and/or the distributions were not normal, they were transformed logarithmically. The transformations were successful in rendering the variance of the observation constant and the distribution of data normal, and hence the transformed values were used for subsequent statistical evaluations. The data were analyzed by one-way ANOVA to establish whether the effect of naringenin was significant, and significant differences of the means at a level of p < 0.05 were evaluated by two-sided Tukey’s test.
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RESULTS Activity and mRNA Levels of Hepatic Lipogenic Enzymes. Naringenin at dietary levels of 1 and 2.5 g/kg did not affect the intake of food during the experiment (Table 1). In addition, no significant differences were seen in growth, body weight, and liver weight at the time of killing among the groups. Table 1. Effect of Dietary Naringenin on Growth Parameters and Liver Weighta body wt (g) initial final growth (g/15 d) food intake (g/d) liver wt (g/100 g body wt) a
0b
1b
2.5b
128 ± 2 260 ± 5 132 ± 3 19.3 ± 0.9 5.45 ± 0.17
128 ± 3 257 ± 2 130 ± 4 19.8 ± 0.5 5.49 ± 0.15
127 ± 2 259 ± 5 132 ± 5 20.4 ± 0.5 5.48 ± 0.17
Values are means ± SEM, n = 7. bDietary naringenin (g/kg).
A diet containing 2.5 g/kg naringenin compared with a diet free of this flavonoid caused a significant 62% decrease (p = 0.0002) in the activity of fatty acid synthase (Figure 1). Although the decreases were attenuated, naringenin at this dietary level caused significant 24, 21, and 31% decreases in the activities of ATP-citrate lyase, 6-phosphogluconate dehydrogenase, and pyruvate kinase, respectively (p-values were 0.029, 0.042, and 0.010, respectively). Glucose 6-phosphate dehydrogenase activity was 20% lower in rats fed a diet containing 2.5 g/kg naringenin than in the animals fed a diet free of this flavonoid, but the difference was not significant (p = 0.248). The diet containing 1 g/kg naringenin compared to the naringenin-free diet also caused a significant (p = 0.047) 22% decrease in the activity of ATP-citrate lyase. The activities of other enzymes were 9−18% lower in rats fed the diet containing 1 g/kg naringenin than in the animals fed the naringenin-free diet. However, these differences were not significant. Figure 2 shows the mRNA levels of proteins related to lipogenesis. There are two types of acetyl-CoA carboxylase, namely, α and β. The α but not the β form appears to be involved in fatty acid synthesis in the cytosol.32 There are four isoforms of pyruvate kinase in mammals. L-Pyruvate kinase is an enzyme highly expressed in the liver.33 Adiponutrin34 and spot 1435 are proteins assumed to be involved in the regulation of lipogenesis. Naringenin at a dietary level of 2.5 g/kg strongly decreased the mRNA levels of acetyl-CoA carboxylase α, fatty acid synthase, and ATP-citrate lyase (39, 60 and 37% decreases, respectively). The reductions were all significant (p-values were 0.0011, 0.0013, and 0.0011, respectively). Although the reductions were attenuated, the flavonoid at this dietary level also caused significant 23 and 27% decreases, respectively, in the levels of glucose 6-phosphate dehydrogenase and L-pyruvate 9537
DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
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Figure 1. Effect of naringenin on the activity levels of hepatic lipogenic enzymes. Values are the means with their standard errors (n = 7). Means without a common letter differ at p < 0.05.
Figure 2. Effect of naringenin on the mRNA levels of hepatic lipogenic enzymes, adiponutrin, spot 14, and SREBP-1c in rat liver. mRNA levels represent the relative values, setting those in rats fed a control diet free of naringenin as 1. Values are the means with their standard errors (n = 7). Means without a common letter differ at p < 0.05.
2.5 g/kg naringenin (45 and 43% decreases, respectively). The reductions were significant at p-values of 0.0001 and 0.0040, respectively). Naringenin at a dietary level of 2.5 g/kg also
kinase (p-values were 0.029 and 0.0076, respectively). In addition, strong reductions in the mRNA levels of spot 14 and adiponutrin were observed in the animals fed a diet containing 9538
DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
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Journal of Agricultural and Food Chemistry Table 2. Effect of Dietary Naringenin on the Activities of Hepatic Fatty Acid Oxidation Enzymesa enzyme activity (nmol/min per mg protein) peroxisomal palmitoyl-CoA oxidation Acyl-CoA oxidase carnitine palmitoyltransferase enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase 3-ketoacyl-CoA thiolase 2,4-dienoyl-CoA reductase a
0b
1b
2.5b
1.33 ± 0.04 0.952 ± 0.024 2.52 ± 0.11a 4557 ± 128 250 ± 15 144 ± 7 4.13 ± 0.43
1.44 ± 0.12 0.917 ± 0.042 2.46 ± 0.11a 4746 ± 129 240 ± 11 147 ± 8 4.44 ± 0.35
1.37 ± 0.15 0.968 ± 0.071 3.07 ± 0.18b 4887 ± 266 284 ± 15 167 ± 9 4.45 ± 0.46
Values are means ± SEM, n = 7. Means in a row without a common letter differ at p < 0.05. bDietary naringenin (g/kg).
Table 3. Effect of Dietary Naringenin on the Concentrations of Serum and Liver Componentsa 0b serum components triacylglycerol (mmol/L) cholesterol (mmol/L) phospholipid (mmol/L) glucose (mmol/L) insulin (μg/L) naringenin (μmol/L) liver components triacylglycerol (μmol/g) cholesterol (μmol/g) phospholipid (μmol/g) naringenin (μmol/g) a
1b
2.84 ± 0.41 b 3.16 ± 0.13 b 3.64 ± 0.14 b 9.94 ± 0.38 12.7 ± 2.0 0.0 ± 0.0 a
2.28 2.63 3.18 8.98 11.7 13.7
± ± ± ± ± ±
52.5 ± 9.0 b 5.99 ± 0.37 b 37.1 ± 0.7 0.000 ± 0.000 a
24.3 ± 2.4 a 4.53 ± 0.21 a 36.5 ± 0.9 0.174 ± 0.025 b
0.25 ab 0.11 a 0.08 a 0.23 1.7 2.0 b
2.5b 1.72 2.48 2.94 9.19 10.0 27.9
± ± ± ± ± ±
0.17 a 0.13 a 0.14 a 0.18 0.9 3.6 c
20.2 ± 2.1 a 4.27 ± 0.18 a 37.4 ± 0.9 0.367 ± 0.052 c
Values are means ± SEM, n = 7. Means in a row without a common letter differ at p < 0.05. bDietary naringenin (g/kg).
significantly (p = 0.011) decreased the mRNA expression of SREBP-1c (41% decrease) involved in regulation of the mRNA expression of many lipogenic enzymes. Although the diet containing 1 g/kg naringenin was less effective in decreasing mRNA levels of various lipogenic enzymes and of spot 14, adiponutrin, and SREBP-1c, the values were lower or tended to be lower in the animals fed this diet than in the animals fed the diet without this compound. Significant decreases with this diet were observed for mRNA levels of fatty acid synthase, Lpyruvate kinase, and spot 14 (p-values were 0.034, 0.041, and 0.0013, respectively). We also measured mRNA levels of SREBP-2 and of its downstream genes. Naringenin did not affect the mRNA level of SREBP-2 (1.00 ± 7, 1.05 ± 4, and 0.987 ± 0.031 for rats fed naringenin-free and 1 and 2.5 g/kg naringenin diets, respectively). This flavonoid also did not affect mRNA levels of 3-hydroxy-3-metylglutaryl-CoA (HMG-CoA) synthase (1.00 ± 0.12, 0.831 ± 0.134, and 0.827 ± 0.063, respectively), HMGCoA reductase (1.00 ± 0.10, 0.888 ± 0.065, and 0.975 ± 0.075, respectively), or low-density lipoprotein receptor (1.00 ± 0.06, 1.09 ± 0.09, and 0.918 ± 0.055, respectively). The activities of enzymes involved in fatty acid oxidation were also analyzed (Table 2). A diet containing 2.5 g/kg naringenin, but not a diet containing 1 g/kg of naringenin relative to a diet free of this flavonoid, caused a significant 22% increase (p = 0.03) in the activity of carnitine palmitoyltransferase. However, naringenin at dietary levels of 1 and 2.5 g/kg failed to affect the activities of other enzymes. Serum and Liver Lipid and Naringenin Levels. The serum concentration of triacylglycerol in rats fed a diet containing 2.5 g/kg naringenin was about 40% of that observed in animals fed a diet free of naringenin (Table 3). The reduction was significant at p = 0.044. In addition, this
compound at this dietary level caused significant reductions in the serum concentrations of cholesterol (21% decrease) and phospholipid (19% decrease) at p-values of 0.0033 and 0.0025, respectively. Naringenin at the dietary level of 1 g/kg also caused significant decreases in the serum concentrations of cholesterol (17% decrease) and phospholipid (13% decrease) at p-values of 0.021 and 0.046, respectively, but the change observed with serum triacylglycerol (20% decrease) was not significant (p = 0.54). Dietary naringenin even at 1 g/kg significantly decreased the hepatic concentration of triacylglycerol (54% decrease) at a p-value of 0.0024. An additional decrease was observed with a diet containing 2.5 g/kg naringenin (62% decrease). The reduction was significant at a p-value of 0.0002. The hepatic concentrations of cholesterol were significantly lower in rats fed diets containing 1 and 2.5 g/ kg naringenin than in the animals fed a diet free of this flavonoid (24 and 29% decreases, respectively) at p-values of 0.0030 and 0.0007, respectively. Naringenin did not affect the hepatic phospholipid level. Naringenin was detected in the serum and liver of rats fed diets containing 1 and 2.5 g/kg naringenin, but not in the animals fed a diet free of this compound. Naringenin concentrations in serum and liver of rats fed a diet containing 2.5 g/kg naringenin were approximately double those of animals fed a diet containing 1 g/kg of this flavonoid.
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DISCUSSION We employed 15 d of treatment to observe the physiological activity of naringenin in the present study. This was considerably shorter than periods employed in other studies13−21 (3 weeks to 6 months) to evaluate the effect of this flavonoid on lipid metabolism. We previously observed that 7 to 10 d of feeding is sufficient to clearly observe the effect of 9539
DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
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Journal of Agricultural and Food Chemistry
Therefore, it is possible that dietary naringenin reduced the mRNA expressions of lipogenic enzymes through downregulation of the SREBP-1 signaling pathway. SREBP-1c is synthesized as a membrane-bound precursor. To be active, this needed to be cleaved by a two-step proteolytic process in Golgi apparatus to release its amino-terminal region which contains the DNA-binding domain. The mature form of this transcription factor enters the nucleus and activates the genes involved in lipogenesis. Therefore, analysis of the protein content of the mature form of SREBP-1c in the nucleus is required to come to the definite conclusion that naringenin reduces hepatic lipogenesis through the SREBP-1c signaling pathway. Although naringenin did not affect the serum concentration of insulin in the present study, it is possible that this hormone is involved in the naringenin-dependent decrease of hepatic fatty acid synthesis. In fact, it has been well-demonstrated that insulin affects the processing of the precursor form of SREBP-1 to the mature form through alteration of the expression of insulin-induced gene 1 and 2 proteins (insig1 and 2, respectively).40,41 Therefore, naringenin-dependent changes in the insulin signaling pathway should be examined in detail in future studies. In addition to SREBP-1, liver X receptor α (LXRα) plays a crucial role in regulating lipogenesis through interaction with promoters of the genes of SREBP-1c42 and fatty acid synthase.43 In relation to this, Goldwasser et al.38 reported that naringenin caused apparent dose-dependent inhibition of LXRα activity in Huh7 human hepatoma cells at concentrations ranging from 40 to 400 μM. We observed in the present study that the hepatic concentration of naringenin in rats fed this flavonoid at a dietary level of 2.5 g/kg was as high as 0.367 μmol/g. This implies that the concentration of naringenin exceeded 300 μM in the liver in this situation. This is sufficient to cause a strong decrease in LXRα activity. Therefore, it is possible that a naringenin-dependent decrease in LXRα activity is involved in the reduction in hepatic lipogenesis observed here. This needs to be clarified in a future study. We16 previously demonstrated that naringenin at a dietary level of 10 g/kg increased the hepatic activities of various enzymes involved in fatty acid oxidation, accompanying the increases in mRNA levels of peroxisomal fatty acid oxidation enzymes in ICR mice. In relation to this, Goldwasser et al.38 reported that naringenin at concentrations ranging from 2.4 to 240 μM dose-dependently activated peroxisome proliferator activated receptor α (PPARα), a transcription factor involved in the regulation of fatty acid oxidation enzymes. In addition, Cho et al.21 reported that dietary naringenin, even at low dietary levels (0.03 to 0.12 g/kg), dose-dependently increased the protein expression of PPARα and its target genes (carnitine palmitoyltransferase 1 and uncoupling protein 2) in the liver of Long-Evans hooded rats. In the present study, dietary naringenin caused a significant increase in the activities of carnitine palmitoyltransferase, but not in the other enzymes. Therefore, it is rather difficult to conclude that naringenin increased hepatic fatty acid oxidation, at least in Sprague− Dawley rats, in spite of the fact that the hepatic concentration of naringenin in rats fed this flavonoid appeared to be high enough to cause strong activation of PPARα.38 Some studies13−15 indicated that naringenin lowered the activity of HMG-CoA reductase, a rate-limiting enzyme in cholesterol biosynthesis, in rats fed a high-cholesterol diet. So, it is possible that naringenin not only downregulates the SREBP-
dietary factors on hepatic lipogenesis and fatty acid oxidation.36,37 The extension of the experimental period did not necessarily enhance the physiological activity of food factors affecting these metabolic processes.36 So, it is conceivable that 15 d treatment is long enough to observe the physiological activity of naringenin affecting hepatic fatty acid synthesis and oxidation. Felgineset al.30 measured the plasma concentration of naringenin in rats meal-fed a diet containing 2.5 g/kg naringenin (25 g diet/rat) to clarify the availability of this flavonoid. The plasma naringenin concentration reached a peak 10 h after completing the meal-feeding and decreased thereafter. The peak value exceeded 120 μmol/L. The value observed 24 after the meal-feeding (about 20 μmol/L) appeared comparable to that observed in the present study in rats fed a 2.5 g/kg naringenin diet ad libitum. They reported that about 28% of naringenin was recovered in urine within 24 h after a single meal, which indicated that the absorbability of this flavonoid exceeds 28%. No information regarding the tissue concentration of naringenin in rats fed this flavonoid is available. The values observed in the present study appear high enough to affect various signaling pathways.38 Although these observations supported the view that bioavailability of dietary naringenin is high enough to exhibit its meritorious effects, trials to improve the bioaccessibility would still be required to potentiate the physiological activity of this flavonoid supplied in the diet.39 Some studies have indicated that dietary naringenin decreases hepatic fatty acid synthesis in C57BL/KsJ-db/db mice17 and C57BL/6J LDL receptor-null mice fed a Western diet.19 This study has provided the first evidence that this flavonoid is also effective in reducing hepatic lipogenesis in Sprague−Dawley rats fed a purified diet containing saturated fat (palm oil). As a diet containing 2.5 g/kg naringenin caused significant decreases in the activities of various hepatic lipogenic enzymes and mRNA levels of various proteins related to the regulation of lipogenesis, it is clear that this flavonoid has potential to reduce hepatic lipogenesis not only in mice but also in rats. The dietary levels of this flavonoid required to observe downregulation of hepatic lipogenesis appeared to differ considerably among these studies. Jung et al.17 reported that naringenin at a dietary level as low as 0.2 g/kg caused significant 26% and 28% decreases, respectively, in the activities of fatty acid synthase and glucose 6-phosphate dehydrogenase in C57BL/KsJ-db/db mice. Mulvihillet al.,19 employing a much higher dietary level (30 g/kg), observed that this flavonoid caused about a 25% decrease in hepatic lipogenesis, estimated in vivo using [14C]acetic acid as a tracer, and about a 50% decrease in the mRNA level of SREBP-1c. In the present study, a dietary level of 2.5 g/kg was required to observe apparent decreases in parameters of lipogenesis. Therefore, it is plausible that the effectiveness of this flavonoid in altering hepatic lipogenesis depends on various factors, including the animal species, the genetic background, and the type of experimental diet. SREBP-1c is a transcription factor that regulates the expression of genes involved hepatic lipogenesis. Among the various proteins that we analyzed regarding their mRNA levels, acetyl-CoA carboxylase α, fatty acid synthase, ATP-citrate lyase, glucose 6-phosphate dehydrogenase, malic enzyme, pyruvate kinase, spot 14, and SREBP-1 itself have been demonstrated to be under the control of this transcription factor.40,41 Dietary naringenin reduced the mRNA expressions of all these proteins. 9540
DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
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Journal of Agricultural and Food Chemistry Notes
1 signaling pathway but also affects the SREBP-2 signaling pathway and hence reduces cholesterol synthesis which, in turn, causes the lowering of cholesterol levels in the serum and liver. However, naringenin did not affect mRNA levels of SREBP-2 or its downstream genes including cholesterogenic enzymes in the present study. Naringenin may not affect hepatic cholesterol biosynthesis, at least in Sprague−Dawley rats fed cholesterol-free diets. Therefore, a reduction of cholesterol synthesis cannot account for the cholesterol-lowering effect of naringenin observed in the present study. Dietary naringenin decreased the serum concentrations of triacylglycerol, cholesterol, and phospholipid and the hepatic concentrations of triacylglycerol and cholesterol. The decrease in hepatic fatty acid synthesis may account for the lipidlowering effect of this flavonoid. However, in spite of the fact that the decreases in parameters of fatty acid synthesis were much stronger with a diet containing 2.5 g/kg naringenin than with a diet containing 1 g/kg of this flavonoid, the decreases in serum and liver lipids were comparable between these two diets. In relation to this, studies have indicated that naringenin affects various aspects of lipid metabolism including apoB secretion and microsomal triglyceride transfer protein expression.44−46 In addition, studies indicated that some plant flavonoids lower mRNA expression of diacylglycerol acyltransferase in the liver of rats47 and in HepG2 cells.48 Therefore, not only the alterations of fatty acid synthesis but also those of other metabolic processes in the liver may contribute to naringenin-dependent changes in lipid profiles in the serum and liver. The results of the present and previous studies13−22 showed that the citrus flavonoid naringenin has a potent hypolipidemic effect in rats and mice. In relation to this, several epidemiological studies49−51 showed that there is an inverse correlation between fruit and vegetable consumption and the risk of atherosclerosis. Some of this effect may be ascribed to the consumption of naringenin. In conclusion, we found that dietary naringenin at a dietary level of 2.5 g/kg significantly decreased the activity and mRNA levels of various lipogenic enzymes in the liver of Sprague− Dawley rats. Although the effects were considerably attenuated, a diet containing 1 g/kg of this flavonoid also significantly lowered some of these parameters. Therefore, it is apparent that naringenin has potential to reduce hepatic lipogenesis in rats. Downregulation of hepatic lipogenesis may be a crucial factor for the lipid-lowering effect of dietary naringenin. Clarification of the exact molecular mechanisms underlying the naringenindependent decrease of hepatic lipogenesis is required in future studies.
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The authors declare no competing financial interest.
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AUTHOR INFORMATION
Corresponding Author
*Department of Food and Nutrition, Faculty of Human Life, Jumonji University, 2-1-28 Sugasawa, Niiza, Saitama 352-8510, Japan. Tel: 81-48-260-7620. Fax: 81-48-478-9367. E-mail:
[email protected]. Present Address ‡
T.H.: Nihon Waters K. K., No. 5 Koike Building, 1-3-12 Kitashinagawa, Shinagawa-ku, Tokyo 140-0001, Japan. Funding
This study was supported in part by a grant-in-aid for scientific research (Scientific Research C, No. 25450177) from the Japan Society for the Promotion of Science. 9541
DOI: 10.1021/acs.jafc.5b03734 J. Agric. Food Chem. 2015, 63, 9536−9542
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